Rat pheochromocytoma PC12 cells and bovine adrenal chromaffin cells are used to study the mechanism of secretion and its regulation by Ca2+ and GTP-binding proteins. Our goals are to determine how Ca2+ induces secretion and to identify some of the proteins involved in the final steps of the secretory process. Pertussis toxin-modification of either bovine chromaffin cells or PC12 cells results in both a decrease in cytoskeletal F-actin and an increase in secretory activity. These effects of pertussis toxin appear to be independent of changes in second messengers. We have found that pertussis toxin-modification of unstimulated PC12 cells results in about a 50% inhibition of protein phosphatase 2A, a major cellular serine/threonine- specific protein phosphatase. This decrease in phosphatase activity appears to occur in the absence of any extracellular agonist and could account for both the increase in secretory activity and the decrease in cytoskeletal F-actin. Because phosphorylation regulates the activities of many cell surface receptors and/or their intracellular targets, a pertussis toxin-induced decrease in protein phosphatase 2A activity could alter signaling pathways in which G-proteins are not directly involved. Secretion of norepinephrine by digitonin-permeabilized PC12 cells can be stimulated by the addition of GTPgammaS in the absence of Ca2+. GTPgammaS also stimulates Ca2+-independent secretion in a number of other types of cells. As in PCl2 cells, these stimulations appear to be independent of any known second messenger. The class of GTP-binding proteins responsible for these stimulations has been referred to as Ge, for exocytosis. Thus far, no Ge has been isolated. We have isolated from bovine brain membranes a mixture of low molecular weight GTP binding proteins which increases GTPgammaS-stimulated secretion by digitonin-permeabilized PC12 cells. We are currently working to identify which low molecular GTP- binding protein is responsible for this stimulation.